Genetic diversity of Cryptosporidium identified in clinical samples from cities in Brazil and Argentina

The identification and characterisation of Cryptosporidiumgenotypes and subtypes are fundamental to the study of cryptosporidiosis epidemiology, aiding in prevention and control strategies. The objective was to determine the genetic diversity ofCryptosporidium in samples obtained from hospitals of Rio de Janeiro, Brazil, and Buenos Aires, Argentina. Samples were analysed by microscopy and TaqMan polymerase chain reaction (PCR) assays forCryptosporidium detection, genotyped by nested-PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene and subtyped by DNA sequencing of the gp60 gene. Among the 89 samples from Rio de Janeiro, Cryptosporidium spp were detected in 26 by microscopy/TaqMan PCR. In samples from Buenos Aires,Cryptosporidium was diagnosed in 15 patients of the 132 studied. The TaqMan PCR and the nested-PCR-RFLP detected Cryptosporidium parvum, Cryptosporidium hominis, and co-infections of both species. In Brazilian samples, the subtypes IbA10G2 and IIcA5G3 were observed. The subtypes found in Argentinean samples were IbA10G2, IaA10G1R4, IaA11G1R4, and IeA11G3T3, and mixed subtypes of Ia and IIa families were detected in the co-infections. C. hominis was the species more frequently detected, and subtype family Ib was reported in both countries. Subtype diversity was higher in Buenos Aires than in Rio de Janeiro and two new subtypes were described for the first time.     

Molecular epidemiology of cryptosporidiosis: An update

Molecular tools have been developed to detect and differentiate Cryptosporidium at the species/genotype and subtype levels. These tools have been increasingly used in characterizing the transmission of Cryptosporidium spp. in humans and animals. Results of these molecular epidemiologic studies have led to better appreciation of the public health importance of Cryptosporidium species/genotypes in various animals and improved understanding of infection sources in humans. Geographic, seasonal and socioeconomic differences in the distribution of Cryptosporidium spp. in humans have been identified, and have been attributed to differences in infection sources and transmission routes. The transmission of C. parvum in humans is mostly anthroponotic in developing countries, with zoonotic infections play an important role in developed countries. Species of Cryptosporidium and subtype families of C. hominis have been shown to induce different clinical manifestations and have different potential to cause outbreaks. The wide use of a new generation of genotyping and subtyping tools in well designed epidemiologic studies should lead to a more in-depth understanding of the epidemiology of cryptosporidiosis in humans and animals.     

Multiplex-Touchdown PCR to Simultaneously Detect Cryptosporidium parvum,Giardia lamblia,and Cyclospora cayetanensis,the Major Causes of Traveler’s Diarrhea

This study aimed to develop a multiplex-touchdown PCR method to simultaneously detect 3 species of protozoan parasites, i.e., Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis, the major causes of traveler’s diarrhea and are resistant to standard antimicrobial treatments. The target genes included the Cryptosporidium oocyst wall protein for C. parvum, Glutamate dehydrogenase for G. lamblia, and 18S ribosomal RNA (18S rRNA) for C. cayetanensis. The sizes of the amplified fragments were 555, 188, and 400 bps, respectively. The multiplex-touchdown PCR protocol using a primer mixture simultaneously detected protozoa in human stools, and the amplified gene was detected in >1×10³ oocysts for C. parvum, >1×10⁴ cysts for G. lamblia, and >1 copy of the 18S rRNA gene for C. cayetanensis. Taken together, our protocol convincingly demonstrated the ability to simultaneously detect C. parvum, G. lamblia, and C. cayetanenesis in stool samples.     

Genetic richness and diversity in Cryptosporidium hominis and C. parvum reveals major knowledge gaps and a need for the application of “next generation” technologies — Research review

Cryptosporidium species (apicomplexan protists) are a major cause of diarrhoeal disease (= cryptosporidiosis) in humans worldwide. The impact of cryptosporidiosis is also compounded by the spread of HIV/AIDS and a lack of cost-effective anti-cryptosporidial chemotherapeutics or vaccines. Mitigation of the impact of cryptosporidiosis in humans needs to focus on prevention and control strategies, built on a sound understanding of the epidemiology of Cryptosporidium species. Refined epidemiological studies rely on the use of molecular tools employing informative genetic markers. Currently, the 60-kDa glycoprotein gene (gp60) is the most suitable and widely used genetic marker for Cryptosporidium species infecting humans. Here, we undertake an analysis of all publicly-available gp60 sequence data and associated literature for C. hominis and C. parvum, and yield useful insights into the richness, diversity and distribution of genetic variants, and link these variants to human cryptosporidiosis. This global analysis reveals that, despite high genetic richness in Cryptosporidium isolates from humans, there is a surprisingly low diversity. It also highlights limited knowledge about the genetics of cryptosporidiosis in developing nations and in many animals that might act as infection sources. Clearly, there is a major need for more comprehensive studies of Cryptosporidium infecting humans and other animals in Africa and Asia. As molecular technologies improve and become affordable, future studies should utilize “next generation” sequencing and bioinformatic platforms to conduct comparative ‘genome sequence surveys’ to test the validity of current genetic classifications based on gp60 data. Complemented by in vitro and in vivo investigations, these biotechnological advances will also assist significantly in the search for new intervention strategies against human cryptosporidiosis.     

The CpA135 gene as a marker to identify Cryptosporidium species infecting humans

Of the 22 species currently recognized as valid in the Cryptosporidium genus, C. parvum and C. hominis account for most cases of human infections worldwide. However, C. meleagridis, C. canis, C. felis, C. suis, C. muris, as well as the cervine, rabbit and monkey Cryptosporidium genotypes, have also been recognized as the etiologic cause of both sporadic and epidemic cryptosporidiosis in humans. Molecular methods are necessary to distinguish species and genotypes of Cryptosporidium, due to the lack of reliable morphological variations. The aim of this work was to determine the genetic polymorphisms in a fragment of the A135 gene in isolates of C. parvum, C. hominis, C. meleagridis, C. canis, C. muris, C. andersoni and the Cryptosporidium cervine genotype. Primers were designed on conserved regions identified on a multiple alignment of the C. parvum, C. hominis and C. muris sequences, the three species for which information is available at the genome level. PCR amplification and direct sequencing of a 576 bp fragment revealed the presence of numerous single nucleotide polymorphisms (SNPs) among the species/genotype tested. The genetic variability was exploited to design a PCR-RFLP assay useful for a rapid identification of the most important human pathogens in the genus Cryptosporidium.     

Identification of rare and novel Cryptosporidium GP60 subtypes in human isolates from Jordan

Little is known about the epidemiology of Cryptosporidium in Jordan and no genotyping studies have been conducted on Cryptosporidium isolates from humans or animals from Jordan. Genotyping of 44 Cryptosporidium isolates from Jordanian children at the 18S rRNA locus and a unique diagnostic locus identified four Cryptosporidium species; C. parvum (22), C. hominis (20), C. meleagridis (1) and C. canis (1). Sub-genotype analysis of 29 isolates at the 60-kDa glycoprotein (GP60) locus identified three C. parvum, two C. hominis subtype families and one C. meleagridis subtype. Several rare and novel subtypes were identified indicating unique endemicity and transmission of Cryptosporidium in Jordan.     

Comparative genomic analysis reveals occurrence of genetic recombination in virulent Cryptosporidium hominis subtypes and telomeric gene duplications in Cryptosporidium parvum

Background

Cryptosporidium hominis is a dominant species for human cryptosporidiosis. Within the species, IbA10G2 is the most virulent subtype responsible for all C. hominis–associated outbreaks in Europe and Australia, and is a dominant outbreak subtype in the United States. In recent yearsIaA28R4 is becoming a major new subtype in the United States. In this study, we sequenced the genomes of two field specimens from each of the two subtypes and conducted a comparative genomic analysis of the obtained sequences with those from the only fully sequenced Cryptosporidium parvum genome.

Results

Altogether, 8.59-9.05 Mb of Cryptosporidium sequences in 45–767 assembled contigs were obtained from the four specimens, representing 94.36-99.47% coverage of the expected genome. These genomes had complete synteny in gene organization and 96.86-97.0% and 99.72-99.83% nucleotide sequence similarities to the published genomes of C. parvum and C. hominis, respectively. Several major insertions and deletions were seen between C. hominis and C. parvum genomes, involving mostly members of multicopy gene families near telomeres. The four C. hominis genomes were highly similar to each other and divergent from the reference IaA25R3 genome in some highly polymorphic regions. Major sequence differences among the four specimens sequenced in this study were in the 5′ and 3′ ends of chromosome 6 and the gp60 region, largely the result of genetic recombination.

Conclusions

The sequence similarity among specimens of the two dominant outbreak subtypes and genetic recombination in chromosome 6, especially around the putative virulence determinant gp60 region, suggest that genetic recombination plays a potential role in the emergence of hyper-transmissible C. hominis subtypes. The high sequence conservation between C. parvum and C. hominis genomes and significant differences in copy numbers of MEDLE family secreted proteins and insulinase-like proteases indicate that telomeric gene duplications could potentially contribute to host expansion in C. parvum.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1517-1) contains supplementary material, which is available to authorized users.     

Infection with anthroponotic Cryptosporidium parvum does not fully protect the host against a subsequent challenge with C. hominis

Cryptosporidium hominis and Cryptosporidium parvum are the major Cryptosporidium species that infect humans. Earlier studies in gnotobiotic piglets, model susceptible to both, showed that piglets recovered from infection with C. hominis were fully protected against challenge with same species but incompletely protected against C. parvum challenge. In the present study, piglets were infected with C. parvum first, and after recovery were re-challenged with C. parvum or C. hominis. Again, full protection was only observed when piglets were challenged with the homologous parasite strain. Although the two species are genetically/antigenically almost identical, they do not confer complete protection against each other.     

Immunoenzymatic analysis and genetic detection of Cryptosporidium parvum in lambs from Italy

Cryptosporidiosis is a worldwide-diffused protozoan disease causing important economic losses to animal husbandry and livestock production. Additionally, several species/genotypes of Cryptosporidium have a relevant zoonotic potential and ruminants may be important sources of infection for human beings. Nonetheless, in Europe, little is known of the presence of Cryptosporidium in sheep nor on the species/genotypes involved. To obtain information on the occurrence of cryptosporidiosis in lambs and the potential zoonotic role of the Cryptosporidium isolates, one hundred and forty-nine faecal samples individually collected from lambs in central Italy have been examined for the presence of Cryptosporidium. All faecal specimens were processed with a commercial ELISA kit immunoassay and all ELISA-positive samples were further analyzed genetically. Twenty-six ELISA-positive samples scored positive at the PCR and the sequences obtained displayed 100% identity with the zoonotic Cryptosporidum parvum. This work suggests for the first time that lambs in Italy may shed C. parvum, thus representing a potential public health hazard.     

Distribution and Clinical Manifestations of Cryptosporidium Species and Subtypes in HIV/AIDS Patients in Ethiopia

Background

Cryptosporidiosis is an important cause for chronic diarrhea and death in HIV/AIDS patients. Among common Cryptosporidium species in humans, C. parvum is responsible for most zoonotic infections in industrialized nations. Nevertheless, the clinical significance of C. parvum and role of zoonotic transmission in cryptosporidiosis epidemiology in developing countries remain unclear.

Methodology/Principal Findings

In this cross-sectional study, 520 HIV/AIDS patients were examined for Cryptosporidium presence in stool samples using genotyping and subtyping techniques. Altogether, 140 (26.9%) patients were positive for Cryptosporidium spp. by PCR-RFLP analysis of the small subunit rRNA gene, belonging to C. parvum (92 patients), C. hominis (25 patients), C. viatorum (10 patients), C. felis (5 patients), C. meleagridis (3 patients), C. canis (2 patients), C. xiaoi (2 patients), and mixture of C. parvum and C. hominis (1 patient). Sequence analyses of the 60 kDa glycoprotein gene revealed a high genetic diversity within the 82 C. parvum and 19 C. hominis specimens subtyped, including C. parvum zoonotic subtype families IIa (71) and IId (5) and anthroponotic subtype families IIc (2), IIb (1), IIe (1) and If-like (2), and C. hominis subtype families Id (13), Ie (5), and Ib (1). Overall, Cryptosporidium infection was associated with the occurrence of diarrhea and vomiting. Diarrhea was attributable mostly to C. parvum subtype family IIa and C. hominis, whereas vomiting was largely attributable to C. hominis and rare Cryptosporidium species. Calf contact was identified as a significant risk factor for infection with Cryptosporidium spp., especially C. parvum subtype family IIa.

Conclusions/Significance

Results of the study indicate that C. parvum is a major cause of cryptosporidiosis in HIV-positive patients and zoonotic transmission is important in cryptosporidiosis epidemiology in Ethiopia. In addition, they confirm that different Cryptosporidium species and subtypes are linked to different clinical manifestations.     

Comparative Sensitivity of PCR Primer Sets for Detection of Cryptosporidium parvum

Improved methods for detection of Cryptosporidium oocysts in environmental and clinical samples are urgently needed to improve detection of cryptosporidiosis. We compared the sensitivity of 7 PCR primer sets for detection of Cryptosporidium parvum. Each target gene was amplified by PCR or nested PCR with serially diluted DNA extracted from purified C. parvum oocysts. The target genes included Cryptosporidium oocyst wall protein (COWP), small subunit ribosomal RNA (SSU rRNA), and random amplified polymorphic DNA. The detection limit of the PCR method ranged from 10³ to 10⁴ oocysts, and the nested PCR method was able to detect 10⁰ to 10² oocysts. A second-round amplification of target genes showed that the nested primer set specific for the COWP gene proved to be the most sensitive one compared to the other primer sets tested in this study and would therefore be useful for the detection of C. parvum.     

Microarray analysis of the human antibody response to synthetic Cryptosporidium glycopeptides

Glycoproteins expressed by Cryptosporidium parvum are immunogenic in infected individuals but the nature of the epitopes recognised in C. parvum glycoproteins is poorly understood. Since a known immunodominant antigen of Cryptosporidium, the 17 kDa glycoprotein, has previously been shown to bind to lectins that recognise the Tn antigen (GalNAcα1-Ser/Thr-R), a large number of glycopeptides with different Tn valency and presentation were prepared. In addition, glycopeptides were synthesised based on a 40 kDa cryptosporidial antigen, a polymorphic surface glycoprotein with varying numbers of serine residues, to determine the reactivity with sera from C. parvum-infected humans. These glycopeptides and non-glycosylated peptides were used to generate a glycopeptide microarray to allow screening of sera from C. parvum-infected individuals for the presence of IgM and IgG antibodies. IgG but not IgM in sera from C. parvum-infected individuals bound to multivalent Tn antigen epitopes presented on glycopeptides, suggesting that glycoproteins from C. parvum that contain the Tn antigen induce immune responses upon infection. In addition, molecular differences in glycosylated peptides (e.g. substituting Ser for Thr) as well as the site of glycosylation had a pronounced effect on reactivity. Lastly, pooled sera from individuals infected with either Toxoplasma or Plasmodium were also tested against the modified Cryptosporidium peptides and some sera showed specific binding to glycopeptide epitopes. These studies reveal that specific anti-glycopeptide antibodies that recognise the Tn antigen may be useful diagnostically and in defining the roles of parasite glycoconjugates in infections.     

Molecular Characterization of Cryptosporidium Isolates from Humans and Animals in Iran

Isolates of Cryptosporidium spp. from human and animal hosts in Iran were characterized on the basis of both the 18S rRNA gene and the Laxer locus. Three Cryptosporidium species, C. hominis, C. parvum, and C. meleagridis, were recognized, and zoonotically transmitted C. parvum was the predominant species found in humans.     

Molecular characterisation of Cryptosporidium outbreaks in Western and South Australia

Molecular typing at the 18S rRNA and Gp60 loci was conducted on Cryptosporidium-positive stool samples from cases collected during 2007 Western Australian and South Australian outbreaks of cryptosporidiosis. Analysis of 48 Western Australian samples identified that all isolates were C. hominis and were from five different Gp60C. hominis subtype families. The IbA10G2 subtype was most common across all age groups (37/48). In South Australia, analysis of 24 outbreak samples, identified 21 C. hominis isolates, two C. parvum isolates and one sample with both C. hominis and C. parvum. All C. hominis isolates were identified as the IbA10G2 subtype.     

Identification of Cryptosporidium felis in a Cow by Morphologic and Molecular Methods

Apicomplexan Cryptosporidium parasites infect a wide range of vertebrate hosts. While some species are limited to a single host group, such as Cryptosporidium baileyi, which infects chickens, other species of this genus, such as C. parvum, infect a wide range of mammalian species from mice to humans. During an investigation of Cryptosporidium infection in cattle on a farm in northern Poland, we identified an infection caused by C. felis, in addition to known infections with C. muris and C. parvum. This new infection was identified based on the size of the oocysts (mean size, 4.3 ± 0.4 μm; range, 3.5 to 5.0 μm), as well as by analysis of the molecular sequence of the variable region of the small-subunit rRNA. This finding demonstrates the complex host specificity and circulation in the environment of Cryptosporidium species.     

Cryptosporidium spp., Giardia intestinalis,and Enterocytozoon bieneusi in Captive Non-Human Primates in Qinling Mountains

Non-human primates (NHPs) are confirmed as reservoirs of Cryptosporidium spp., Giardia intestinalis, and Enterocytozoon bieneusi. In this study, 197 fresh fecal samples from 8 NHP species in Qinling Mountains, northwestern China, were collected and examined using multilocus sequence typing (MLST) method. The results showed that 35 (17.8%) samples were positive for tested parasites, including Cryptosporidium spp. (3.0%), G. intestinalis (2.0%), and E. bieneusi (12.7%). Cryptosporidium spp. were detected in 6 fecal samples of Macaca mulatta, and were identified as C. parvum (n=1) and C. andersoni (n=5). Subtyping analysis showed Cryptosporidium spp. belonged to the C. andersoni MLST subtype (A4, A4, A4, and A1) and C. parvum 60 kDa glycoprotein (gp60) subtype IId A15G2R1. G. intestinalis assemblage E was detected in 3 M. mulatta and 1 Saimiri sciureus. Intra-variations were observed at the triose phosphate isomerase (tpi), beta giardin (bg), and glutamate dehydrogenase (gdh) loci, with 3, 1, and 2 new subtypes found in respective locus. E. bieneusi was found in Cercopithecus neglectus (25.0%), Papio hamadrayas (16.7%), M. mulatta (16.3%), S. sciureus (10%), and Rhinopithecus roxellana (9.5%), with 5 ribosomal internal transcribed spacer (ITS) genotypes: 2 known genotypes (D and BEB6) and 3 novel genotypes (MH, XH, and BSH). These findings indicated the presence of zoonotic potential of Cryptosporidium spp. and E. bieneusi in NHPs in Qinling Mountains. This is the first report of C. andersoni in NHPs. The present study provided basic information for control of cryptosporidiosis, giardiasis, and microsporidiosis in human and animals in this area.     

Longitudinal multi-locus molecular characterisation of sporadic Australian human clinical cases of cryptosporidiosis from 2005 to 2008

Cryptosporidium is a gastrointestinal parasite that is recognised as a significant cause of non-viral diarrhea in both developing and industrialised countries. In the present study, a longitudinal analysis of 248 faecal specimens from Australian humans with gastrointestinal symptoms from 2005 to 2008 was conducted. Sequence analysis of the 18S rRNA gene locus and the 60 kDa glycoprotein (gp60) gene locus revealed that 195 (78.6%) of the cases were due to infection with Cryptosporidium hominis, 49 (19.8%) with Cryptosporidium parvum and four (1.6%) with Cryptosporidium meleagridis. A total of eight gp60 subtype families were identified; five C. hominis subtype families (Ib, Id, Ie, If and Ig), and two C. parvum subtype families (IIa and IId). The Id subtype family was the most common C. hominis subtype family identified in 45.7% of isolates, followed by the Ig subtype family (30.3%) and the Ib subtype family (20%). The most common C. parvum subtype was IIaA18G3R1, identified in 65.3% of isolates. The more rare zoonotic IId A15G1 subtype was identified in one isolate. Statistical analysis showed that the Id subtype was associated with abdominal pain (p < 0.05) and that in sporadic cryptosporidiosis, children aged 5 and below were 1.91 times and 1.88 times more likely to be infected with subtype Id (RR 1.91; 95% CI, 1.7-2.89; p < 0.05) and Ig (RR 1.88; 95% CI, 1.10-3.24; p < 0.05) compared to children aged 5 and above. A subset of isolates were also analysed at the variable CP47 and MSC6-7 gene loci. Findings from this study suggest that anthroponotic transmission of Cryptosporidium plays a major role in the epidemiology of cryptosporidiosis in Western Australian humans.     

Biological and Genetic Characterization of Cryptosporidium spp. and Giardia duodenalis Isolates from Five Hydrographical Basins in Northern Portugal

To understand the situation of water contamination with Cryptosporidium spp. and Giardia spp. in the northern region of Portugal, we have established a long-term program aimed at pinpointing the sources of surface water and environmental contamination, working with the water-supply industry. Here, we describe the results obtained with raw water samples collected in rivers of the 5 hydrographical basins. A total of 283 samples were analyzed using the Method 1623 EPA, USA. Genetic characterization was performed by PCR and sequencing of genes 18S rRNA of Cryptosporidium spp. and β-giardin of Giardia spp. Infectious stages of the protozoa were detected in 72.8% (206 of 283) of the water samples, with 15.2% (43 of 283) positive for Giardia duodenalis cysts, 9.5% (27 of 283) positive for Cryptosporidium spp. oocysts, and 48.1% (136 of 283) samples positive for both parasites. The most common zoonotic species found were G. duodenalis assemblages A-I, A-II, B, and E genotypes, and Cryptosporidium parvum, Cryptosporidium andersoni, Cryptosporidium hominis, and Cryptosporidium muris. These results suggest that cryptosporidiosis and giardiasis are important public health issues in northern Portugal. To the authors'' knowledge, this is the first report evaluating the concentration of environmental stages of Cryptosporidium and Giardia in raw water samples in the northern region of Portugal.     

Predominant Virulent IbA10G2 Subtype of Cryptosporidium hominis in Human Isolates in Barcelona: A Five-Year Study

Background

Cryptosporidium infection is a worldwide cause of diarrheal disease. To gain insight into the epidemiology of the infection in a certain geographic area, molecular methods are needed to determine the species/genotypes and subtypes.

Methodology/Principal Findings

From 2004 to 2009, 161 cryptosporidiosis cases were detected in two hospitals in Barcelona. Diagnosis was performed by microscopic observation of oocysts in stool specimens following modified Ziehl-Neelsen staining. Most cases (82%) occurred in children. The number of cases increased in summer and autumn. Molecular characterization of Cryptosporidium was performed in 69 specimens, and C. hominis and C. parvum were identified in 88.4% and 10.1% of the cases, respectively. C. meleagridis was detected in one specimen. Subtyping based on the gp60 polymorphism showed six subtypes, four C. hominis and two C. parvum. Subtype IbA10G2 was the most prevalent subtype corresponding to 90% of all C. hominis isolates. This is the first report on the distribution of specific Cryptosporidium subtypes from humans in Spain.

Conclusions/Significance

In our geographic area, the anthroponotic behavior of C. hominis, the lower infective dose, and the higher virulence of certain subtypes may contribute to the high incidence of human cryptosporidiosis caused by the IbA10G2 subtype. Further studies should include populations with asymptomatic shedding of the parasite.     

Molecular Characterization of Cryptosporidium Oocysts in Samples of Raw Surface Water and Wastewater

Recent molecular characterizations of Cryptosporidium parasites make it possible to differentiate the human-pathogenic Cryptosporidium parasites from those that do not infect humans and to track the source of Cryptosporidium oocyst contamination in the environment. In this study, we used a small-subunit rRNA-based PCR-restriction fragment length polymorphism (RFLP) technique to detect and characterize Cryptosporidium oocysts in 55 samples of raw surface water collected from several areas in the United States and 49 samples of raw wastewater collected from Milwaukee, Wis. Cryptosporidium parasites were detected in 25 surface water samples and 12 raw wastewater samples. C. parvum human and bovine genotypes were the dominant Cryptosporidium parasites in the surface water samples from sites where there was potential contamination by humans and cattle, whereas C. andersoni was the most common parasite in wastewater. There may be geographic differences in the distribution of Cryptosporidium genotypes in surface water. The PCR-RFLP technique can be a useful alternative method for detection and differentiation of Cryptosporidium parasites in water.